The Strong Anti-Kinetoplastid Properties of Bee Propolis: Composition and Identification of the Active Agents and Their Biochemical Targets

The kinetoplastids are protozoa characterized by the presence of a distinctive organelle, called the kinetoplast, which contains a large amount of DNA (kinetoplast DNA (kDNA)) inside their single mitochondrion. Kinetoplastids of medical and veterinary importance include Trypanosoma spp. (the causative agents of human and animal African Trypanosomiasis and of Chagas disease) and Leishmania spp. (the causative agents of the various forms of leishmaniasis). These neglected diseases affect millions of people across the globe, but drug treatment is hampered by the challenges of toxicity and drug resistance, among others. Propolis (a natural product made by bees) and compounds isolated from it are now being investigated as novel treatments of kinetoplastid infections. The anti-kinetoplastid efficacy of propolis is probably a consequence of its reported activity against kinetoplastid parasites of bees. This article presents a review of the reported anti-kinetoplastid potential of propolis, highlighting its anti-kinetoplastid activity in vitro and in vivo regardless of geographical origin. The mode of action of propolis depends on the organism it is acting on and includes growth inhibition, immunomodulation, macrophage activation, perturbation of the cell membrane architecture, phospholipid disturbances, and mitochondrial targets. This gives ample scope for further investigations toward the rational development of sustainable anti-kinetoplastid drugs

New sulphated flavonoids from Wissadula periplocifolia (L.) C. Presl (Malvaceae)

Wissadula periplocifolia (L.) C. Presl (Malvaceae) is commonly used in Brazil to treat bee stings and as an antiseptic. The antioxidant properties of its extracts have been previously demonstrated, thus justifying a phytochemical investigation for its bioactive phenolic constituents. This has yielded five new sulphated flavonoids: 8-O-sulphate isoscutellarein (yannin) (1a); 4'-O-methyl-7-O-sulphate isoscutellarein (beltraonin) (1b); 7-O-sulphate acacetin (wissadulin) (2a); 4'-O-methyl-8-O-sulphate isoscutellarein (caicoine) (2b) and 3'-O-methyl-8-O-sulphate hypolaetin (pedroin) (3b) along with the known flavonoids 7,4'-di-O-methyl-8-O-sulphate isoscutellarein (4), acacetin, apigenin, isoscutellarein, 4´-O-methyl isoscutellarein, 7,4'-di-O-methylisoscutellarein, astragalin and tiliroside. The compounds were isolated by column chromatography and identified by NMR (¹H, (13)C, HMQC, HMBC and COSY) and LC-HRMS. A cell based assay was carried out to evaluate the preliminary cytotoxic properties of the flavonoids against UVW glioma and PC-3M prostate cancer cells as well as non-tumour cell lines. The obtained results showed that acacetin, tiliroside, a mixture of acacetin + apigenin and the sulphated flavonoids 2a + 2b exhibited inhibitory activity against at least one of the cell lines tested. Among the tested flavonoids acacetin and tiliroside showed lower IC50 values, presenting promising antitumor effects

Activity of Compounds from Temperate Propolis against Trypanosoma brucei and Leishmania mexicana

Ethanolic extracts of samples of temperate zone propolis, four from the UK and one from Poland, were tested against three strains and displayed EC values < 20 µg/mL. The extracts were fractionated, from which 12 compounds and one two-component mixture were isolated, and characterized by NMR and high-resolution mass spectrometry, as 3-acetoxypinobanksin, tectochrysin, kaempferol, pinocembrin, 4'-methoxykaempferol, galangin, chrysin, apigenin, pinostrobin, cinnamic acid, coumaric acid, cinnamyl ester/coumaric acid benzyl ester (mixture), 4',7-dimethoxykaempferol, and naringenin 4',7-dimethyl ether. The isolated compounds were tested against drug-sensitive and drug-resistant strains of and , with the highest activities ≤ 15 µM. The most active compounds against were naringenin 4',7 dimethyl ether and 4'methoxy kaempferol with activity of 15-20 µM against the three strains. The most active compounds against were 4',7-dimethoxykaempferol and the coumaric acid ester mixture, with EC values of 12.9 ± 3.7 µM and 13.1 ± 1.0 µM. No loss of activity was found with the diamidine- and arsenical-resistant or phenanthridine-resistant strains, or the miltefosine-resistant strain; no clear structure activity relationship was observed for the isolated compounds. Temperate propolis yields multiple compounds with anti-kinetoplastid activity

European propolis is highly active against trypanosomatids including Crithidia fasciculata.

Extracts of 35 samples of European propolis were tested against wild type and resistant strains of the protozoal pathogens Trypanosoma brucei, Trypanosoma congolense and Leishmania mexicana. The extracts were also tested against Crithidia fasciculata a close relative of Crithidia mellificae, a parasite of bees. Crithidia, Trypanosoma and Leishmania are all members of the order Kinetoplastida. High levels of activity were obtained for all the samples with the levels of activity varying across the sample set. The highest levels of activity were found against L. mexicana. The propolis samples were profiled by using liquid chromatography with high resolution mass spectrometry (LC-MS) and principal components analysis (PCA) of the data obtained indicated there was a wide variation in the composition of the propolis samples. Orthogonal partial least squares (OPLS) associated a butyrate ester of pinobanksin with high activity against T. brucei whereas in the case of T. congolense high activity was associated with methyl ethers of chrysin and pinobanksin. In the case of C. fasciculata highest activity was associated with methyl ethers of galangin and pinobanksin. OPLS modelling of the activities against L. mexicana using the mass spectrometry produced a less successful model suggesting a wider range of active components

Isolation of a novel flavanonol and an alkylresorcinol with highly potent anti-trypanosomal activity from Libyan propolis

Twelve propolis samples from different parts of Libya were investigated for their phytochemical constituents. Ethanol extracts of the samples and some purified compounds were tested against Trypanosoma brucei, Plasmodium falciparum and against two helminth species, Trichinella spiralis and Caenorhabditis elegans, showing various degrees of activity. Fourteen compounds were isolated from the propolis samples, including a novel compound Taxifolin-3-acetyl-4’-methyl ether (4), a flavanonol derivative. The crude extracts showed moderate activity against T. spiralis and C. elegans, while the purified compounds had low activity against P. falciparum. Anti-trypanosomal activity (EC50 = 0.7 µg/mL) was exhibited by a fraction containing a cardol identified as bilobol (10) and this fraction had no effect on Human Foreskin Fibroblasts (HFF), even at 2.0 mg/mL, thus demonstrating excellent selectivity. A metabolomics study was used to explore the mechanism of action of the fraction and it revealed significant disturbances in trypanosomal phospholipid metabolism, especially the formation of choline phospholipids. We conclude that a potent and highly selective new trypanocide may be present in the fraction

The chemical characterization of Nigerian propolis samples and their activity against Trypanosoma brucei.

Profiling of extracts from twelve propolis samples collected from eight regions in Nigeria was carried out using high performance liquid chromatography (LC) coupled with evaporative light scattering (ELSD), ultraviolet detection (UV) and mass spectrometry (MS), gas chromatography mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (NMR). Principal component analysis (PCA) of the processed LC-MS data demonstrated the varying chemical composition of the samples. Most of the samples were active against Trypanosoma b.brucei with the highest activity being in the samples from Southern Nigeria. The more active samples were fractionated in order to isolate the component(s) responsible for their activity using medium pressure liquid chromatography (MPLC). Three xanthones, 1,3,7-trihydroxy-2,8-di-(3-methylbut-2-enyl)xanthone, 1,3,7-trihydroxy-4,8-di-(3-methylbut-2-enyl)xanthone a previously undescribed xanthone and three triterpenes: ambonic acid, mangiferonic acid and a mixture of α-amyrin with mangiferonic acid (1:3) were isolated and characterised by NMR and LC-MS. These compounds all displayed strong inhibitory activity against T.b.brucei but none of them had higher activity than the crude extracts. Partial least squares (PLS) modelling of the anti-trypanosomal activity of the sample extracts using the LC-MS data indicated that high activity in the extracts, as judged from LCMS 2data, could be correlated to denticulatain isomers in the extracts

Chemical and antimicrobial profiling of propolis from different regions within Libya.

Extracts from twelve samples of propolis collected from different regions of Libya were tested for their activity against Trypanosoma brucei, Leishmania donovani, Plasmodium falciparum, Crithidia fasciculata and Mycobacterium marinum and the cytotoxicity of the extracts was tested against mammalian cells. All the extracts were active to some degree against all of the protozoa and the mycobacterium, exhibiting a range of EC50 values between 1.65 and 53.6 μg/ml. The toxicity against mammalian cell lines was only moderate; the most active extract against the protozoan species, P2, displayed an IC50 value of 53.2 μg/ml. The extracts were profiled by using liquid chromatography coupled to high resolution mass spectrometry. The data sets were extracted using m/z Mine and the accurate masses of the features extracted were searched against the Dictionary of Natural Products (DNP). A principal component analysis (PCA) model was constructed which, in combination with hierarchical cluster analysis (HCA), divided the samples into five groups. The outlying groups had different sets of dominant compounds in the extracts, which could be characterised by their elemental composition. Orthogonal partial least squares (OPLS) analysis was used to link the activity of each extract against the different micro-organisms to particular components in the extracts

Libyan Propolis, a comprehensive chemical, in vitro biological investigation and metabolomic profiling of antiprotozoal activity

Propolis (bee-glue) is collected by bees from plants as a defensive substance in response to environmental pressures which include a range of microorganisms and parasites. These parasites are known to include the protozoal species Crithidia. Since it is collected by bees for the specific purpose of providing chemotherapeutic protection this increases the likelihood of finding active compounds in propolis compared with random screening of plants.;Twelve samples of Libyan propolis (P1-P12) were collected from different geographic zones of Libya. Ethanolic extracts of the twelve propolis samples were prepared and these were profiled initially by NMR which gave some general indication of the type of compounds which might be found in them providing signals typical of diterpene aldehydes and cycloartane triterpenoids depending on the origin of the sample.;There were limited signals in the aromatic region between 6 and 8 ppm in contrast to Northern European samples where many signals from flavonoid compounds would be expected. The extracts were profiled by high resolution LC-MS and the LC-MS data was extracted and modelled by SIMCA-P software using PCA with HCA, which separated the samples into five main groups based on their chemical composition.;The groups were according to Geographic origin which the samples from North East, North West, South East and Southwest Libya grouping together. The sample extracts were tested against a wide range of microorganisms including T. brucei, L. donovani, P. falciparum, C. fasiculata, M. marinum, S. aureus, K. pneumoniae and T. spiralis. In addition, cell based assays for cytotoxicity and anti-inflammatory activities were carried out.;Eighteen isolated compounds were isolated including: eight diterpenes (1) 13-epi-torulosal, (2) 13-O- acetyl epi-cupressic acid, (3) 13-epicupressic acid, (4) 13-epitorulosol, (15) acetylisocuppressic acid, (16) Agathadiol, (17) Isocupressic acid and, (18) isoagatholal, three lignans; (5) sesamin, (6) Demethylpiperitol, (7) 5', methoxy piperitol, (8) the flavonoid flavanone taxifolin-3-acetate-4'-methyl ether and five triterpenes of the cyclo artane type; (9) cycloartanol, (10) mangiferolic acid, (11) mangiferonic acid, (12) ambolic acid, (13) 27-hydroxymangeferonic acid and the resorcinol (14) cardol.;Both the crude extracts and isolated compounds exhibited activity against the range of microorganisms were tested such as T. brucei, L. donovani, P. falciparum, C. fasiculata, M. marinum, S. aureus, K. pneumoniae and T. spiralis.Propolis (bee-glue) is collected by bees from plants as a defensive substance in response to environmental pressures which include a range of microorganisms and parasites. These parasites are known to include the protozoal species Crithidia. Since it is collected by bees for the specific purpose of providing chemotherapeutic protection this increases the likelihood of finding active compounds in propolis compared with random screening of plants.;Twelve samples of Libyan propolis (P1-P12) were collected from different geographic zones of Libya. Ethanolic extracts of the twelve propolis samples were prepared and these were profiled initially by NMR which gave some general indication of the type of compounds which might be found in them providing signals typical of diterpene aldehydes and cycloartane triterpenoids depending on the origin of the sample.;There were limited signals in the aromatic region between 6 and 8 ppm in contrast to Northern European samples where many signals from flavonoid compounds would be expected. The extracts were profiled by high resolution LC-MS and the LC-MS data was extracted and modelled by SIMCA-P software using PCA with HCA, which separated the samples into five main groups based on their chemical composition.;The groups were according to Geographic origin which the samples from North East, North West, South East and Southwest Libya grouping together. The sample extracts were tested against a wide range of microorganisms including T. brucei, L. donovani, P. falciparum, C. fasiculata, M. marinum, S. aureus, K. pneumoniae and T. spiralis. In addition, cell based assays for cytotoxicity and anti-inflammatory activities were carried out.;Eighteen isolated compounds were isolated including: eight diterpenes (1) 13-epi-torulosal, (2) 13-O- acetyl epi-cupressic acid, (3) 13-epicupressic acid, (4) 13-epitorulosol, (15) acetylisocuppressic acid, (16) Agathadiol, (17) Isocupressic acid and, (18) isoagatholal, three lignans; (5) sesamin, (6) Demethylpiperitol, (7) 5', methoxy piperitol, (8) the flavonoid flavanone taxifolin-3-acetate-4'-methyl ether and five triterpenes of the cyclo artane type; (9) cycloartanol, (10) mangiferolic acid, (11) mangiferonic acid, (12) ambolic acid, (13) 27-hydroxymangeferonic acid and the resorcinol (14) cardol.;Both the crude extracts and isolated compounds exhibited activity against the range of microorganisms were tested such as T. brucei, L. donovani, P. falciparum, C. fasiculata, M. marinum, S. aureus, K. pneumoniae and T. spiralis

Chromatographic analysis with different detectors in the chemical characterisation and dereplication of African propolis

Propolis or bee glue has very diverse composition and is potentially a source of biologically active compounds. Comprehensive chemical profiling was performed on 22 African propolis samples collected from the sub-Saharan region of Africa by using various hyphenated analytical techniques including Liquid Chromatography (LC)-UltraViolet Detection (UV)-Evaporative Light Scattering Detection (ELSD), LC-High Resolution Mass Spectrometry (HRMS), Gas Chromatography (GC)-MS and LC-Diode Array Detector (DAD)-HRMS/MS. The diversity of the composition of these African propolis samples could be observed by heat mapping the LC-UV and ELSD data. The characteristic chemical components were uncovered by applying Principal Component Analysis (PCA) to the LC-HRMS data and a preliminary dereplication was carried out by searching their accurate masses in the Dictionary of Natural Products (DNP). A further identification was achieved by comparing their GC-MS or LC-DAD-HRMS/MS spectra with previously published data. Generally no clear geographic delineation was observed in the classification of these African propolis samples. Triterpenoids were found as the major chemical components in more than half of the propolis samples analysed in this study and some others were classified as temperate and Eastern Mediterranean type of propolis. Based on the comparative chemical profiling and dereplication studies one uncommon propolis from southern Nigeria stood out from others by presenting prenylated isoflavonoids, which indicated that it was more like Brazilian red propolis, and more significantly a high abundance of stilbenoid compounds which could be novel in propolis